Short-term in vitro and in vivo genotoxicity testing systems for some ...

Environ Monit Assess (2011) 180:87–95 DOI 10.1007/s10661-010-1774-z

Short-term in vitro and in vivo genotoxicity testing systems for some water bodies of Northern India Athar Habib Siddiqui · Shams Tabrez · Masood Ahmad

Received: 3 May 2010 / Accepted: 1 November 2010 / Published online: 1 December 2010 © Springer Science+Business Media B.V. 2010

Abstract The genotoxicity of certain water bodies was evaluated employing the DNA repair defective mutants of Escherichia coli, induction of prophage lamda in the lysogen and the plasmid nicking assay. All the test DNA repair defective mutants invariably exhibited more sensitivity than their isogenic wild-type strains but distinctive patterns against the three water samples viz. industrial waste water and the groundwater samples obtained from industrial estate of Aligarh as well as river water of Yamuna at Agra. A significant level of phage induction was also recorded in the test system exhibiting maximum induction in case of industrial waste water followed by that in river and groundwater samples, respectively. The single- and double-strand breaks were also observed in the plasmid DNA treated with in-

Electronic supplementary material The online version of this article (doi:10.1007/s10661-010-1774-z) contains supplementary material, which is available to authorized users. A. H. Siddiqui · S. Tabrez · M. Ahmad (B) Department of Biochemistry, Faculty of Life Sciences, AMU, Aligarh, 202002, India e-mail: [email protected], [email protected]

dustrial waste water and the river water samples. These findings are suggestive of the DNA damage induced by the test samples with the probable role of SOS repair in E. coli. Keywords Genotoxicity · Water bodies · SOS repair · Prophage induction · Plasmid nicking assay

Introduction DNA damage in higher living organisms is the major cause of cancer and a host of other diseases (Maru and Bhide 1989; Zhou and Elledge 2000). Various short-term bioassays have, therefore, been extensively used to monitor the genotoxicity of environmental pollutants (Dutka 1996; Fatima and Ahmad 2006). DNA damage has also been proposed to be a useful parameter in screening chemicals for their genotoxic properties, since many chemical carcinogens and mutagens have been shown to induce DNA damage in mammalian cells (Ewig and Kohn 1977; Schwarz et al. 1979; Pastink et al. 2001). Moreover, DNA strand breakage and the formation of deoxyribonucleoside chemical adducts have proven to be very helpful in assaying the DNA damaging potential of various chemicals under in vitro conditions (Lutz 1979; Azuma et al. 2001)

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Environ Monit Assess (2011) 180:87–95

Among the various bacterial genotoxicity tests, DNA repair assays take a prominent position (Quillardet et al. 1982; Kerklaan et al. 1985; Hellmer and Boldsfoldi 1992; Muller and Janz 1992). These repair assays usually estimate the extent of DNA damage to determine the genotoxic/mutagenic potential of a chemical or test system. The induction of SOS repair system in bacteria following exposure to DNA damaging agents is another critical parameter for genotoxicity testing (Bagg et al. 1981; Gottesman 1981; Kenyon and Walker 1981). A high correlation between DNA repair system in bacteria and higher organisms has also been well established (Leifer et al. 1981; d’Ari 1985; Brazmanova et al. 2001). The work undertaken in this study affirms the feasibility of one in vitro system i.e. plasmid nicking assay and two in vivo bioassays, namely the survival pattern of Escherichia coli K-12 repairdefective mutants and the λ-prophage induction test for the evaluation of genotoxic potential of various water bodies of India.

Materials and methods Media and strains The media and their components for the growth of E. coli K-12 strains and the λ-lysogen were obtained from Hi-Media (India). The genetic markers associated with various E. coli strains used in this study are given in Table 1. Water sampling and sample concentration procedures Water sampling procedure from river was strictly in accordance with APHA (1998). The industrial wastewater and groundwater samples were the composite samples taken from five different sites within a region of 15 km2 for each experiment. The groundwater samples were collected from the hand pumps used by the local population which pump out the water at a depth of not more than 30 ft. The groundwater and the river water

Table 1 The characteristics of E. coli K-12 strains and the λ-lysogen used in the study Strain designation AB 1157

AB1886

AB2463

AB2480 AB2494

JW164 JW165 λc1857

Relevant genetic markers

Source qsr1− ,

Thr-1, araC14, leu B6, (gpt-proA)62, lacY1, tsx-33, glnV44(AS), λ− , Rac-0, hisG4(Oc), rfbD1, mgl-51, rpoS396(Am), rpsL31(strR), kdgK51, xylA5, mtl-1, arg E3(Oc), thi-1 Thr-1, araC14, leu B6, (gpt-proA) 62, lacY1, tsx-33, qsr1−, glnV44(AS), galK2(Oc), λ− , Rac-0, hisG4(Oc), rfbD1, mgl-51, rpoS396(Am), rpsL31(strR), kdgK51, xylA5, mtl-1, arg E3(Oc), thi-1, uvrA6 Thr-1, araC14, leu B6, (gpt-proA) 62, lacY1, tsx-33, qsr1-, glnV44(AS), galK2(Oc), λ-, Rac-0, hisG4(Oc), rfbD1, rpsL31(strR), kdgK51, xylA5, mtl-1, arg E3(Oc), thi-1, recA13 (gpt-proA)62, lacY1, tsx-33?, glnV44(AS)?, galK2(Oc), λ-, recA13, rpsL31(strR), or rpsl8,xylA5, mtl-1, thi-1, uvrA6 Thr-1, araC14, leu B6, (gpt-proA) 62, lacY1, tsx-33, glnV44(AS), galK2(Oc), λ-, Rac-0?, his G4(Oc), rfbD1?, mgl-51?, rpsL31(strR), kdgK51?, xylA5, mtl-1, metB1, thi-1, lexA1 lacZ53(Am, λ-), thyA36, IN(rrnD-rrnE)1, rpsL151(strR), rha-5, malB45, deoC2 lacZ53(Am, λ-, thyA36, IN(rrnD-rrnE)1, rpsL151(strR), rha-5, malB45, deoC2, polA1(Am), λc1857(ts), relA1, spoT1, thi-1

Dr. Mary, K. Berlyn Yale University U.S.A.

–do–

–do–

–do– –do–

–do– –do– –do–


samples were subjected to 300-fold concentration using XAD-8 resin as described by Wilcox and Williamson (1986). The extraction of the concentrated pollutants eluted from the column was carried out using DMSO. The column was always pretreated with the extracting solvent, followed by thorough washing by distilled water for equilibration.

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counts/ml). The pellets so obtained were resuspended in 10 mM MgSO4 solution. These suspensions were then treated with an equal volume of the test water samples. Aliquots were withdrawn at regular intervals of 2 up to 6 h, suitably diluted and plated to assay the colony forming ability. Plates were incubated over night (O/N) at 37◦ C. For concentrated water samples, the solvent control was also run simultaneously.

Brief description of the sampling area The sampling area for the industrial and groundwater samples houses several electroplating and lock manufacturing small industries within the area of 15 km2 with a moderate population density of labour class. The groundwater samples were presumably containing the pollutants of industrial wastesvia percolation into the shallow aquifer. The river water samples were obtained from the Yamuna, the second largest river of India and were collected from the downstream of Agra, supposedly loaded with the municipal wastes and the industrial effluents. Plasmid nicking assay This assay was carried out as described by Rahman et al. (1990). Covalently closed circular plasmid pBR322 DNA was obtained from Bangalore Genei (India); 0.5 μg plasmid DNA in a final volume of 30 μl was treated with the test water samples for 3 h. After the treatment, 8 μl of ×5 tracking dye (40 mM EDTA, 0.05% bromophenol blue and 50% (v/v) glycerol) was added and loaded in 1% agarose gel. The gel was run at 50 mA for 2 h and stained with ethidium bromide (0.5 μg/l) for 30 min at 4◦ C. After washing, the bands were visualised on photodyne UVtransilluminator (USA) and photographed. Treatment of SOS-defective E. coli K-12 mutants The survival of SOS-defective mutants was determined following the established procedure (Rehana et al. 1995). The cells of various SOSdefective mutants and the isogenic wild-type strains were harvested by centrifugation from exponentially growing culture (1.5 × 108 viable

λ−Prophage induction assay in the test-water samples The method of Qadri and Ahmad (1994) was followed for the phage induction test. Exponentially growing lysogen of λcI857 (1.4 × 108 viable cells/ml) was centrifuged, washed and resuspended in 10 mM MgSO4 solutions. The resuspended cells were treated with the test water samples at 50% concentration (equal volumes of 10 mM MgSO4 solution and test water samples) at 32◦ C for 3 h. The treated cells were again centrifuged, washed and resuspended in nutrient broth with and without chloramphenicol (100 μg/ml) and incubated at 32◦ C for the next 3 h. Aliquots were taken out at regular intervals, suitably diluted and plated with AB1157 cells. The plaques were scored after O/N incubation at 42◦ C. The solvent control was also run simultaneously.

Results Plasmid nicking assay with industrial waste water and river water The industrial waste water and the XAD extracted river water samples brought about the conversion of a significant proportion of supercoiled DNA to relaxed and linear forms (Fig. 1a). The lane ‘a’ in Fig. 1a shows the band patterns of untreated pBR322 DNA to serve as a control. The lanes ‘b’ and ‘c’ indicate the electrophoretic pattern of the of DNA treated with 10 μl of industrial waste water sample and ×300 XAD concentrated river water sample (equivalent to 3 ml of the actual sample) respectively. As is evident from the bands in lane ‘b’, the industrial

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Fig. 1 a Electrophoretic pattern of pBR322 DNA treated with industrial waste water and XAD concentrated river water samples. b Electrophoretic pattern of pBR322 DNA treated with industrial waste water and XAD concentrated river water samples

waste water transformed the supercoiled form of the DNA to both the open circle (nicked form) and the linear form whereas the band observed in lane ‘c’ corresponds to the open circle form of the plasmid DNA which, shows the effect on plasmid following treatments with the river water samples. An increase in the treatment dose (i.e. 20 μl sample) resulted in further damage. The bands in lane ‘b’ of Fig. 1b shows smearing, apparently caused by the degradation of plasmid DNA treated with the 20 μl industrial waste water. The bands in lane ‘c’ of Fig. 1b showing the 20 μl river water treated plasmid DNA reveal the transformation of the supercoiled form of the plasmid DNA to

the open circle (nicked form) and the linear form. These findings are indicative of the fact that these water samples could bring about the damage to the naked DNA molecules.

Fig. 2 Survival of E. coli K-12 DNA repair-defective mutants subjected to industrial wastewater treatment

Fig. 3 Survival of E. coli K-12 DNA repair-defective mutants subjected to direct Yamuna river water treatment

Survival pattern of E. coli K-12 strains exposed to different contaminated water samples Survival of E. coli strains treated with industrial waste water of Aligarh city The survival profile of the E. coli K-12 strains subjected to industrial wastewater treatment is shown in Fig. 2. The pollutants in the wastewater


Fig. 4 Survival of E. coli K-12 DNA repair-defective mutants subjected to XAD-extracted groundwater treatment

samples brought about a significant decline in the colony forming ability of mutants compared to their wild-type counterparts. The strain AB2480 carrying both the uvrA as well as the recA mutation showed the maximum sensitivity followed by that of AB1886, the uvrA single mutant. The survivals of uvrArecA double mutant, uvrA mutant and recA mutant at 6 h treatment were 0.01%, 0.52% and 1%, respectively. The isogenic wildtype AB1157 strain and the lexA mutant on the other hand exhibited 40% and 4.2% survival, respectively. However, with the same exposure, the polA mutant, JW165 and its isogenic wild type, JW164 strain showed 4.2% and 22% survival, respectively. The order of sensitivity of the DNA

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Fig. 5 Survival of E. coli K-12 DNA repair defective mutants subjected to XAD-extracted Yamuna water treatment

repair-defective mutants upon treatment with this water sample could be sequenced as: uvrArecA > uvrA > recA > lexA ≥ polA

Survival of E. coli K-12 strains treated with river water samples from the Yamuna (without concentration) Survival pattern of the various E. coli strains subjected to the river water treatment is presented in Fig. 3. Maximum sensitivity was observed with the uvrArecA double mutant at 6 h treatment (4.2%) compared to its isogenic wild-type which showed 52% survival. The recA, uvrA, lexA and polA

Table 2 λ-Prophage induction by industrial waste water of Aligarh city and river water sample of Yamuna Time of incubation of

Fold induction (with respect to untreated lysogen)

lysogen in LB at 32◦ C (h)

λ-lysogen suspended in buffer for 3 h (negative control)

After treatment with test water samples of Aligarh for 3 h (1:1 diluted)

After treatment with test water samples of Yamuna for 3 h (1:1 diluted)

0 1 2 3

1.0 1.1 1.7 1.9

1.0 3.2 16.7 22.2

1.0 1.0 1.9 2.5

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Table 3 λ-Prophage induction by XAD extracts of groundwater and Yamuna river water samples Time of incubation of lysogen in LB at 32◦ C (h)

Fold induction (with respect to untreated lysogen) Solvent control XAD-extracted Solvent control DMSO groundwater sample (×300)

XAD-extracted Yamuna river water (×300)

0 1 2 3

1.0 1.0 1.0 1.1

1.0 2.8 5.8 10.3

mutants can be grouped in order of sensitivity as under: uvr ArecA > recA > uvrA > lexA > polA Survival of E. coli K-12 strains treated with ×300 concentrated groundwater samples The direct groundwater samples did not show a significant decline in the viability up to 6 h of treatment. However, the survival pattern observed with concentrated samples was quite interesting. The polA strain was found to be the most sensitive (0.81%) compared to recA (1.63%) and lexA (2.7%). The survival pattern is shown in Fig. 4. The order of sensitivity of the test DNA repair defective mutants is shown as under: polA ≥ recA > lexA Survival of E. coli K-12 strains treated with ×300 XAD-extracted river water of Yamuna at Agra Figure 5 shows the survival patterns of E. coli K-12 strains incubated at various time intervals with Yamuna river water samples. The recA mutant displayed the maximum sensitivity (1.09%) followed it closely was the polA mutant (1.66%). The lexA was observed to be the least sensitive mutant (3.6%) in this case. The sensitivity of the different strains under the experimental condition was recorded to be: recA > polA > lexA λ−Prophage induction in the lysogen treated with test water samples The level of induction of λ-prophage as a result of treatment to the lysogen with various water

1.0 1.6 2.8 3.2

1.0 1.0 1.0 1.0

samples has been presented in Tables 2 and 3. A fraction of the lysogenic population exhibited induction of lytic cycle during the liquid holding for 3 h at 32◦ C. Moreover, it appeared that extent of induction was quite high compared with that of control. There was an insignificant increase in the prophage induction over and above the background level in the untreated solvent control (Tables 2 and 3) as well as in the chloramphenicol supplemented tubes containing the test water samples (data not shown). The industrial waste water brought about the maximum induction of lysogen and following it was that of the ×300 XAD concentrated sample of the Yamuna river at Agra. Surprisingly, even the direct water of the Yamuna river also showed a moderate increase in the PFU, suggesting thereby the presence of significant levels of genotoxicants in this water samples. The concentrated groundwater samples obtained from the industrial area of Aligarh city could also bring about a significant level of prophage induction in the lysogen.

Discussion The ability of an organism to survive in an environment specifically damaging to its DNA is attributed to a variety of inherent repair mechanisms (Modrich 1991; Zhou and Elledge 2000). When the repair pathways are blocked and the cellular DNA is left unrepaired, it can result in cell death and mutation (Leadon 1996; Pastink et al. 2001; Peltomaki 2001). In the bacterium E. coli, DNA damage or stalled DNA replication triggers a set of functions collectively called the SOS response. The regula-


tion of the SOS response involves a repressor, the LexA protein, and an inducer, the RecA protein. After DNA is damaged, an effector molecule is produced, possibly a single-stranded DNA which activates the RecA protein to a form, catalysing the proteolytic cleavage of LexA protein. Since the LexA protein represses many genes including those involved in repair function, the proteolytic cleavage of LexA protein under SOS conditions results in the enhanced expression of the recA+ and lexA+ genes also (d’Ari 1985). The repressors of certain bacteriophages like that of λc1857 are also cleaved under the same conditions, resulting in the induction of lytic cycle in lysogen. In general, the substances that are genotoxic to higher eukaryotes also induce the SOS response in bacteria. This correlation is the basis of the numerous bacterial tests for genotoxicity and carcinogenicity (Maron and Ames 1983). The recA, lexA and polA mutants were found to be highly sensitive to treatment with the test water samples (Figs. 2, 3, 4 and 5) suggesting, thereby, the damage to the DNA of the exposed cells as well as the role of recA+ , lexA+ and polA+ genes to cope with their hazardous effect. The role of these genes in the SOS repair of E. coli K-12 is well documented (Walker 1985; Courcelle et al. 2001; Coureclle and Hanawalt 2001; Alam et al. 2009). This idea gains further support from the induction of prophage in the λ-lysogen (Tables 2 and 3). Prophage induction by radiation, chemicals and other mutagenic compounds has been well established and is considered to be one among the many SOS responses (De Marini et al. 1994; Cabrera 2000; Vargas et al. 2001). The efficacy of the E. coli K-12 repair defective mutants in assaying the genotoxicity of surface water and industrial waste has been established in our lab (ISGE 1990; Malik and Ahmad 1995; Rehana et al. 1996). Rehana et al. (1996) in their studies have reported the sensitivity of recA and polA mutants in the evaluation of the genotoxicity of the Ganges water samples. Our results are consistent with the previous studies establishing the validity of this system in the genotoxicity determination of complex environmental mixtures. Moreover, the suitability of prophage induction test in assaying the genotoxicity of riverine water samples has also been established by Vargas et al. (2001).

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With regard to the relative damage caused by the test water samples on various E. coli mutants, the recA strain was usually found to be the most sensitive followed by the lexA and polA strains. The polA mutant on the other hand was comparatively more sensitive in case of the XAD concentrated water samples. These findings are suggestive of the different composition of genotoxicants in the two test systems. The industrial waste water without any concentration brought about the maximum prophage induction and thus was the most toxic sample. Groundwater samples, however, could not bring about a significant level of SOS induction per se. Concentration of the pollutants could obviously be increased by the XAD extraction procedure. This test is, therefore, an important tool to detect the genotoxicity of water samples by XAD concentration method when the mutagens are supposed to be present in low amounts. Supercoiled plasmid DNA is a common substrate for rapid and sensitive assay of the reactions inducing nicks (Rahman et al. 1990). The conversion of the covalently closed circular plasmid DNA to the nicked and linear forms is evident in our system, which further establishes the DNA damaging potential of the water samples under study (Fig. 1a, b).

Conclusions The in vivo bioassays along with the in vitro test confirm the validity of these short-term assays for evaluating the genotoxic activity of water samples. E. coli DNA repair tests have proven to be superior for detecting direct acting agents. Moreover, our results are consistent with the idea that the test water samples initiate the SOS responses that could bring about the mutation in bacterial DNA under in vivo conditions. Furthermore, the test water samples could also induce the damage in the naked DNA as is evident from the plasmid nicking assay. These systems could also serve as preliminary carcinogenicity testing system for the test samples/compounds. In view of the high sensitivity and simplicity, we recommend that these systems may also be

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included in the battery of toxicity testing system along with the popular bioassays. Acknowledgements The authors gratefully acknowledge the financial assistance to the department by the UGC, New Delhi under its DRS programme.

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